Diagnostic compositions for glutamic oxalic transaminase (got) and glutamic pyruvic transaminase (gpt) determinations

ABSTRACT

Stable, lyophilized diagnostic compositions are disclosed effective in determining glutamic oxalic transaminase (GOT) and glutamic pyruvic transaminase (GPT) comprising malate dehydrogenase (for GOT) or lactate dehydrogenase (for GPT), a pyridine nucleotide and a buffer having a sodium content of less than about 600 meq./1. capable of regulating the pH of the composition in reconstituted form. Also disclosed are the aqueous reconstituted products, the method of determining the activity of GOT and GPT in a sample using the subject compositions and the process for making the disclosed diagnostic compositions.

United States Patent [19] Rush et a1.

1 1 DIAGNOSTIC COMPOSITIONS FOR GLUTAMIC OXALIC TRANSAMINASE (GOT) ANDGLUTAMIC PYRUVIC TRANSAMINASE (GPT) DETERMINATIONS [75] Inventors:Robert L. Rush, Spring Valley; Luis P. Leon, Ossining; Anne C. De Lea,Yonkers, all of NY.

[73] Assignee: Technicon Instruments Corporation,

Tarrytown, NY.

[22] Filed: July 20, 1971 [21] Appl. No.: 164,415

[52] US. Cl. 195/103.5 R, 195/99 [51] Int. Cl. GOln 31/14 [58] Field ofSearch 195/103.5 R, 150

[451 June 25, 1974 OTHER PUBLICATIONS Bergmeyer, Methods of EnzymaticAnalysis pp.

Stable, lyophilized diagnostic compositions are disclosed effectiveindetermining glutamic oxalic transaminase (GOT) and glutamic pyruvictransaminase (GPT) comprising malate dehydrogenase (for GOT) or lactatedehydrogenase (for GPT), a pyridine nucleotide and a buffer having asodium content of less than about 600 meq./ 1. capable of regulating thepH of the composition in reconstituted form. Also disclosed are theaqueous reconstituted products, the method of determining the activityof GOT and GPT in a sample using the subject compositions and theprocess for making the disclosed diagnostic compositions.

46 Claims, N0 Drawings DIAGNOSTIC COMPOSITIONS FOR GLUTAlVflC OXALICTRANSAMINASE (GOT) AND GLUTAMIC PYRUVIC TRANSAMINASE (GPT)DETERMINATIONS BACKGROUND OF THE INVENTION This invention relatesgenerally to stable, lyophilized diagnostic compositions. Moreparticularly, it relates to unusually stable novel diagnosticcompositions useful in determining the activity of glutamic oxalictransaminase (GOT) and glutamic pyruvic transaminase (GP'T) in samplescontaining said enzymes.

GOT and GPT are enzymes whose elevated presence in the bloodstream ofhumans and animals is indicative of certain malfunctions and/ordisorders. For example, elevated levels of glutamic oxalic transaminaseis an important indication of the status of patients with severe heartdisease. Accordingly, the determination of the activity of this enzymein the body is also a useful diagnostic tool for doctors and hospitalpersonnel.

Generally, if any heart muscle damage has taken place or is occurring,this will manifest itself as an increase in GOT levels. In addition tosignaling heart disease, elevated GOT levels also indicate thepossibility of severe liver disfunctions such as cirrhosis, cancer ofthe liver or hepatitis.

Similarly, the elevated presence of GPT is indicative of liverdisfunctions of the kind just named. Consequently, the prognosis andcause of liver disease can be determined by an accurate GPTdetermination.

It is readily apparent that certain characteristics are essential for amaterial to be an effective diagnostic reagent. These include, amongothers, good stability, ease of manufacture and consistency incomposition. Diagnostic reagents are commonly prepared and used in thereconstituted state i.e., as an aqueous solution. It is unusual toobserve any substantial degree of stability in diagnostic reagents inthis form.

For this reason, the operator will generally prepare the reagent justprior to use by simply combining the necessary components. The drawbacksof this manipulation are glaring: it is subject to human error; it istime consuming; standardization of the reagentis required; and mostimportantly, production of a consistent reagent is virtually impossible.

The presently disclosed compositions overcome these severe handicaps,the most crucial being the stability aspect. When preparing a reagent asdescribed above, it is imperative that the reagent be used within arelatively short period of time. If not, decomposition which takes placerenders the material totally useless and it must be discarded.

It is one object of this invention to prepare stable, lyophilizeddiagnostic compositions which can be stored for indefinite periods oftime..ln addition, once a batch of material is prepared and a sample isstandardized, that step is no longer necessary and one need only add theproper amount of water for reconstitution. Accordingly, the possibilityof human error is minimized, the time consuming steps of preparationjust prior to use and standardization are eliminated, and mostimportantly, a stable product capable of prolonged storage is availablefor use at any time requiring only a simple re-' constitution step priorto use.

The prior art has not disclosed stable, lyophilized di-' agnosticreagents which are effective in determining GOT and GPT in samplescontaining these enzymes which are free of the abovedescribeddisadvantages.

The present invention does disclose such compositions, their effectiveuse and the means for preparing same.

SUMMARY OF THE INVENTION Accordingly, this invention relates to twostable, lyophilized diagnostic compositions. The first is highlyeffective in determining glutamic oxalic transaminase (GOT) andcomprises malate dehydrogenase (MDH); a pyridine nucleotide such asreduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamideadenine dinucleotide phosphate (NADPH); and a buffer having a sodiumcontent of less than about 600 meq./l. capable of regulating the pH ofthe reconstituted product.

The second is highly effective in determining glutamic pyruvictransaminase (GPT) and comprises lactate dehydrogenase (LDH); a pyridinenucleotide such as reduced nicotinamide adenine dinucleotide (NADH)orreduced nicotinamide adenine dinucleotide phosphate (NADPH); and abuffer having a sodium content of less than about 600 meq./ l. capableof regulating the pH of the reconstituted product.

The reconstituted form of each of these compositions is preferred and isprepared simply by adding a predetermined amount of water resulting in asolution having a pH preferably in the range from 6.0 to 8.0. It ispossible and within the purview of this invention toprepare anintermediate reconstituted product outside the pH range of 6 to 8 whichcan then be adjusted subse quently by adding more buffer thereto.Although the dry reagent can be used in the analytical determination,

it is preferred to use the reconstituted form.

Another preferred embodiment of both compositions concerns the use ofNADH as the pyridine nucleotide component.

The reconstituted forms of these compositions are characterized bycritical properties and limitations which must be present in order tohave a diagnostic reagent having the highly desirable advantagesdescribed earlier. For instance, for the GOT reagent, the MDH must bepresent in an amount to provide an activity of from about 0.5 units/ml.to about 10 units/mL, with a 3 units/ml. activity being the mostpreferred.

.For the GPT reagent, the corresponding LDH activity upon reconstitutionmust be present in an amount to provide activity of from 8 units/ml. toabout units/ml, with a 40 units/ml. activity being most pre ferred.

The amount of pyridine nucleotide, preferable NADH, is present in bothreconstituted compositions in an amount to obtain an optical density inthe range of from 2.1 to 1.0 spectrophotometrically determined at340 nmusing a 10 mm. light path. Most preferred is an optical density of 1.8.

The buffer in both reconstituted compositions has a sodium content lessthan 600 meq./ l. and is capable of regulating the pH of thereconstituted product. The

forms most preferred. If the particular sodium or potassium salt is notspecified, the di-salt is intended.

If sodium phosphate alone is used as the buffer, the amount used isdictated by the sodium content in the reconstituted product. It shouldnot surpass a concentration of 600 meq./l. and is preferably about 400meq./ I.

When potassium phosphate is the buffer, it is added in an amount toprovide a concentration of from 0.05 M to 0.4 M, with a 0.1 Mconcentration most preferred.

The type of buffer which is of choice for purposes of this invention aresalts derived from strong bases and weak acids. Sodium and potassiumphosphate are typical examples. Others of this type include ammoniumacetate, lithium phosphate and sodium borate.

However, other buffers which are not within this category are equallyapplicable. For instance, representative usefulillustrationgnclgdgalbumin and tris buffertris(hydroxymethyl)aminomethane.

Another preferred embodiment of both compositions concerns the inclusionof albumin. The preferred concentration upon reconstitution is about 0.l 5 g./ 100 ml.

Yet another preferred aspect of this invention concerns the use of thesecompositions in the quantitative procedures for determining theparticular enzyme.

In both techniques, the usual procedure consists of mixing the samplewith a substrate and subsequently analyzing for enzyme. This is usuallyeffected in an instrument devised for that purpose. The inventiveembodiment comprises combining the herein disclosed diagnosticcomposition with the reaction product and photometrically measuring thedecrease in NADH concentration.

In the GOT determination, the substrate is preferably an asparticacid-a-ketoglutaric acid mixture whereas in the GPT determination, thesubstrate is preferably an alanine-a-ketoglutaric acid mixture.

The unknown enzyme concentration in both procedures is directlyproportional to the decrease in NADH concentration photometricallydetermined.

Another important embodiment of this invention deals with thepreparation of the stable, lyophilized diagnostic compositions. Theprocess is the same for both and comprises the commingling of MDH or LDHwith pyridine nucleotide and buffer. The resulting aqueous solution islyophilized. The reconstituted products having a pH in the range from6.0 to 8.0 are characterized by the limiting properties described above.

A preferred aspect of both processes concerns the use of potassiumphosphate as buffer and the inclusion of albumin as a reagent.

DETAILED DESCRIPTION OF THE INVENTION should possess an MDH activity offrom 0.5 units/ml.

to about 20 units/ml. in order to provide an efficient diagnosticreagent. An activity of 3 units/ml. is preferred.

The amount of LDH which one adds to the GPT diagnostic reagent iscalculated by determining the activity of a reconstituted product. Forpurposes of this invention, it is found that the reconstituted productshould possess an LDH activity of 8 units/ml. to about 80 units/ml. Anactivity of 40 units/ml is preferred.

The pyridine nucleotide components of the herein disclosed invention arealso commercially available. In preparing the diagnostic compositions,it is mandatory that the NADH or NADPH is in the reduced state since theultimate determination of enzyme in the sample is directly proportionalto the decrease in NADH in the reduced state to the oxidized state,namely, NAD. Similarly, if NADPH is used, the corresponding decrease inreduced state to oxidized state (NADP) is measured.

NADH and NADPH are well known co-factors and are particularly useful inenzymatic reactions requiring oxidation.

The amount of pyridine nucleotide in the diagnostic reagent (for bothGOT and GPT) is determined by measuring the optical density of thereconstituted product. It is found that an optical density in the rangefrom 2.1 to 1.0 spectrophotometrically determined at 340 nm using a 10mm. light path is necessary to provide the effective diagnostic reagentsof this invention.

The third component of the subject compositions, namely, the buffer, isthe most important. It appears to play a critical role in thestabilization of the lyophilized material as well as the reconstitutedproduct. The manner which brings this about is not fully understood atthis time.

With both the GOT and GPT reagents, the type of buffer required for asatisfactory product should have a sodium content of less than 600meq./l. capable of regulating the pH of the composition, preferably inthe range from 6.0 to 8.0. The preferred buffer is potassium phosphateadded in an amount to provide from In both the GOT and GPT diagnosticcompositions,

0.05 M to 0.4 M based on reconstituted material. Most preferred is aconcentration of 0.1 M. Other buffers of preference include sodiumphosphate-potassium phosphate mixtures and sodium phosphate by itself.

It is apparent from the above discussion, that the diagnosticcompositions of this invention are characterized by properties exhibitedin the reconstituted product. Accordingly, the processes for preparingsuch compositions are not critical with respect to initial reagentconcentrations so long as the final product upon reconstitution containsthe well defined properties clearly delineated above.

The process for preparing the subject diagnostic compositions comprisesadmixing MDH or LDH, pyridine nucleotide and buffer material. Theresulting aqueous solution is then lyophilized to provide a dry, stablediagnostic reagent.

If the diagnostic reagent is to contain albumin, it is included as areagent during the mixing step. Although albumin, preferably bovinealbumin, is not an essential component, it serves a highly usefulpurpose. It prevents the settling of solid materials to avoid base-linedrift during the analyzing procedure. However, it plays no role in theenzymatic reaction mechanism. When incorporated into the diagnosticcomposition, it should be present in an amount to provide aconcentration upon reconstitution of about 0.15/ 100 ml. This applies toboth diagnostic compositions, i.e., the GOT and GPT reagents.

At this point, it should be mentioned that the reconstituted diagnosticreagents disclosed herein characterized in the matter described abovewill preferably have a pH in the range of 6.0 to 8.0. If thereconstituted product is outside this range, adjustment thereof will beeffected prior to use in the determination.

In the process for preparing the GOT or GPT reagent, it is preferred touse a 0.5 potassium phosphate buffer solution, a 90 percent by weightaqueous solution of NADH (or NADPH) and an ammonium sulfate suspensionof MDH or LDH.

When bovine albumin is included, it is supplied in the solid state.

The lyophilized compositions of this disclosure are highly stable. Forinstance, they can be stored indefinitely at a temperature of 4C andthey are stable at 45C for at least 2 days.

Even the reconstituted product is relatively stable. It is stable for upto 12 hours at 4C without change in optical density and at 25C, theoptical density changes less than .2 percent in 3 hours.

The subject diagnostic compositions are suitably used in automaticanalyses of samples for GOT and GPT activity. It is known in determiningfor GOT and GPT to mix the sample (under investigation) with a substrateand analyze for enzyme activity. The mixing step may simultaneously beheated. Moreover, itis generally preferred to pass the reaction mixturethrough a dialyzing membrane. The dialysis product is then analyzed forenzyme activity.

The present invention relates in part to the improved modification ofcombining the dialysis product with the GOT or GPT diagnosticcomposition, usually in reconstituted form, and photometricallymeasuring the decrease in NADH concentration.

The concentration of enzyme in question is directly proportional to thedecrease in NADH concentration.

The sample used in the GOT determination can bea serum sample (SGOT), aplasma sample, or it can be an aqueous solution, suspension ordispersion. An example of the latter type is an albumin sample. In theGOT analyses, the preferred substrate is an aspaitic -a-ketoglutaricacid mixture. For the GPT test, an alanine -a-ketoglutaric acid mixtureis the substrate of choice.

EXAMPLE I Preparation of Glutamic Oxalic Transaminase (GOT) DiagnosticComposition To 800 mlaof an 0.5 M potassium phosphate aqueous solutionis added 1.25 g. of NADH (90 percent by weight aqueous solution)followed by the addition of 15,000 units of MDH as an ammonium sulfatesuspension. The resulting solution is brought to 1 liter volume by theaddition of a supplemental amount of.0.5 M aq. potassium phosphatebufferand then dispersed into vials in 5 ml. portions.

The vials are then subjected to lyophilizing conditions and theresulting dry, stable compositions stored at 4C.

RECONSTITUTION The dry compositions prepared above are reconstituted bythe addition of 25 ml. of distilled water. The reconstituted diagnosticreagent exhibits the following properties:

Optical Density 1.75 i 0.05 MDH Activity 3 units/ml. Potassium phosphateconc. 0.1 M

The dry, lyophilized product can be stored indefinitely at a temperatureof 4C and is stable for approximately 2 days at 45C.

The reconstituted product is stable for up to 12 hours at 4C withoutchange in optical density, and at 25C the optical density changes lessthan 0.2 percent in 3 hours.

EXAMPLE ll The procedure of Example I is repeated except 7.5 g.

of bovine albumin is included in the 0.5 M potassium.

phosphate buffer solution.

The properties of the reconstituted product are unchanged and thelyophilized materialhas-comparable stability.

EXAMPLE [II RECONSTITUTION The dry compositions prepared above arereconstitutedby the addition of 25 ml. of distilled water. The

reconstituted diagnostic reagent is characterized by the followingproperties:

Optical Density 1.75 i 0.05 LDH Activity 40 units/ml. Potassiumphosphate conc. 0.1 M

The .dry, lyophilized product can be stored indefinitely ata temperatureof 4C and is stable for at least ,2 days at-45C.

The: reconstituted product is stable for up to 12 hoursat-4Cwithoutchange in optical density,.and at25C the optical densitychanges less than 0.2 percent in 3 hours.

EXAMPLE IV Theprocedure of Example III is repeated except 1.5

.g. of; bovine albumin isincluded in the-800 ml.,portion of 0. 5 Mpotassium .phosphate buffer solution.

The properties of the resulting reconstituted product are unchanged andthe lyophilized material has comparable stability.

EXAMPLE V The reconstituted diagnostic reagent prepared according toExample II is used in determining the GOT activity of an unknown samplein the following manner:

Using AutoAnalyzer II, SMA-l2/60, and SMA-l2 MICRO (all registeredtrademarks of Technicon Instruments Corporation, Tarrytown, N.Y.), thesample (about 0.3 ml.) is mixed with a substrate comprising an asparticacid aketoglutaric acid mixture (75:1 by weight; about 0.4 ml.)dissolved in a phosphate buffer. Theproducts of the reaction dialyzethrough a dialyzer and are reacted with the reconstituted reagent asprepared according to Example II (about 0.3 ml.). The change in opticaldensity is determined in a photometer at 340 nm.

The activity of GOT is directly proportional to the decrease in theoptical density of the NADH.

EXAMPLE VI The procedure of Example V is repeated for the determinationof GPT activity in a sample except the reconstituted diagnostic reagentof Example IV is used in place of the reagent of Example 11 and thesubstrate is an alanine-a-ketoglutaric acid mixture.

EXAMPLE VII The procedure of Example I is repeated to preparereconstituted diagnostic reagents having the following properties:

A B C pH 6 8 7 Optical Density l.() 2.1 1.5 MDH Activity (units/ml.) 0.510 5 Potassium phosphate conc. 0.05M 0.4M 0.3M

EXAMPLE VIII The procedure of Example III is repeated to preparereconstituted diagnostic reagents having the following properties:

The procedure of Example I is repeated except that instead of potassiumphosphate, a buffer consisting of a mixture of potassium phosphate andsodium phosphate is used. The reconstituted product has a sodium contentof 400 meq./ l. and exhibits comparable stability.

EXAMPLE X The procedure of Example I is repeated except that sodiumphosphate buffer is used in place of potassium phosphate. Thereconstituted product has a sodium content of 400 meq./1. and exhibitscomparable activity.

EXAMPLE XI The procedure of Example V is repeated for the determinationof GPT activity of an unknown sample wherein instead of the diagnosticreagent prepared according to Example II, the one described in ExampleIV I is used instead.

What is claimed is:

l. A stable, lyophilized diagnostic composition effective in determiningglutamic oxalic transaminase (GOT) in a sample containing said enzymewhich consists of malate dehydrogenase (MDH) present in an amount toprovide an activity upon reconstitution of from about 0.5 units/ml toabout 10 units/ml; a pyridine nucleotide selected from the groupconsisting of reduced nicotinamide adenine dinucleotide (NADH) andnicotinamide adenine dinucleotide phosphate (NADPH), said pyridinenucleotide present in an amount to obtain an optical density uponreconstitution in the range of from 2.1 to 1.0 spectrophotometricallydetermined at 340 nm using a 10 mm light path; and a buffer selectedfrom the group consisting of potassium phosphate and a mixture ofpotassium phosphate and sodium phosphate, said mixture having a sodiumcontent less than 600 meq./1 upon reconstitution, which regulates the pHof the reconstituted product in the range from 6 to 8.

2. The composition as claimed in claim 1 wherein said buffer ispotassium phosphate present in an amount to provide a concentration offrom about 0.05 M to 0.4 M upon reconstitution.

3. The composition as claimed in claim 1 wherein said pyridinenucleotide is NADH.

4. The composition as claimed in claim 1 wherein said activity of MDH isabout 3 units/ml.

5. The composition as claimed in claim 1 wherein said optical density is1.8.

6. The composition as claimed in claim 2 wherein said. concentration is0.1 M.

7. The composition as claimed in claim 1 wherein said buffer is amixture of potassium phosphate and sodium phosphate and albumin isincluded in an amount to provide a concentration upon reconstitution ofabout 0.15 g./l00 ml.

8. A stable aqueous solution having a pH in the range from 6.0 to 8.0which comprises the composition of claim 1 dissolved in water.

9. A stable, lyophilized diagnostic composition effective in determiningglutamic pyruvic transaminase (GPT) in a sample containing said enzymewhich consists of lactate dehydrogenase (LDH) present in an amount toprovide an activity upon reconstitution of from 8 units/ml to aboutunits/ml; a pyridine nucleotide selected from the group consisting ofreduced nicotinamide adenine dinucleotide (NADH) and reducednicotinamide adenine dinucleotide phosphate (NADPI-I) said pyridinenucleotide present in an amount to obtain an optical density uponreconstitution in the range of from 2.1 to 1.0 spectrophotometricallydetermined at 340 nm using a 10 mm light path; and a buffer selectedfrom the group consisting of potassium phosphate and a mixture ofpotassium phosphate and sodium phosphate, said mixture having a sodiumcontent less than 600 meq./l upon reconstitution. which regulates the pHof the reconstituted product in the range from 6 to 8.

10. The composition as claimed in claim 9 wherein said buffer ispotassium phosphate present in an amount to provide a concentration offrom about 0.05 M to 0.4 M upon reconstitution.

11. The composition as claimed in claim 9 wherein said pyridinenucleotide is NADH.

12. The composition as claimed in claim 9 wherein said activity of LDHis about 40 units/ml.

13. The composition as claimed in claim 9 wherein said optical densityis 1.8.

14. The composition as claimed in claim 10 wherein said concentration is0.1 M.

15. The composition as claimed in claim 9 wherein said buffer is amixture of potassium phosphate and sodium phosphate and albumin isincluded in an amount to provide a concentration upon reconstitution ofabout 0.15 g./l00 ml.

16. A stable aqueous solution having a pH in the range from 6.0 to 8.0which comprises the composition of claim 9 dissolved in water.

17. In a method for quantitatively determining the activity of glutamicoxalic transaminase (GOT) in a sample by mixing said sample and asubstrate and analyzing for GOT; the improvement which comprisescombining the stabilized composition as claimed in claim 1 with thereaction product from said mixing step and photometrically measuring thedecrease in NADH concentration. v

18. The method of claim 17 including the further step of passing thereaction product through a dialysis membrane prior to combining withsaid composition.

19. The method of claim 17 including the further step of heating thereaction mixture of said sample and substrate. V

20. The method of claim 17 wherein said substrate is an aspartic acida-ketoglutaric acid mixture.

21. The method of claim 17 wherein said composition buffer regulates thereconstituted product in the range from 6 to 8.

22. The method of claim 21 wherein said buffer is potassium phosphate.

23. The method of claim 21 wherein said buffer is a mixture of potassiumphosphate and sodium phosphate.

24. The method of claim 22 wherein said composition comprises malatedehydrogenase, NADH and potassium phosphate.

25. The method of claim 17 wherein said composition is in the form of anaqueous solution having a pH in the range from 6.0 to 8.0.

26. In a method for quantitatively determining the activity of glutamicpyruvic transaminase (GPT) in a sample by mixing said sample and asubstrate and analyzing for GPT, the improvement which comprisescombining the stabilized composition as claimed in claim 9 with thereaction product from said mixing step and photometrically measuring thedecrease in NADH concentration.

27. The method of claim 26 including the further step of passing thereaction product of said sample and said substrate through a dialysismembrane prior to combining with said composition.

28. The method of claim 26 including the further step of heating themixture of said sample and substrate.

29. The method of claim 26 wherein said substrate is an alanine-a-ketoglutaric acid mixture.

30. The method of claim 26 wherein said composition buffer regulates thereconstituted product in the range from 6 to 8.

31. The method of claim 30 wherein said buffer is potassium phosphate.

32. The method of claim 30 wherein said buffer is a mixture of potassiumphosphate and sodium phosphate.

33. The method of claim 31 wherein said composition comprises lactatedehydrogenase, NADH and potassium phosphate.

34. The method of claim 26 wherein said composition is in the form of anaqueous solution having a pH in the range from 6.0 to 8.0.

35. A process for the preparation of a stable, lyophilized diagnosticcomposition as claimed in claim I which comprises admixing malatedehydrogenase. a pyridine nucleotide selected from the group consistingof reduced nicotinamide adenine dinucleotide (NADH) and nicotinamideadenine dinucleotide phosphate (NADPH); and a buffer selected from thegroup consisting of potassium phosphate and a mixture ofpotassiumphosphate and sodium phosphate which regulates the pH of thereconstituted product in the range from 6 to 8; and lyophilizing theresulting aqueous product which upon reconstitution is characterized byan optical density within the range from 2.1 to 1.0 and an MDH activityof from 0:5 units/ml. to about 10 units/ml.

36. The process of claim 35 wherein said buffer is potassium phosphateadded as an 0.5 M aqueous solution whose concentration uponreconstitution is from 0.05 to 0.4 M.

37. The process of claim '35 wherein said pyridine nucleotide is NADHand the reconstituted lyophilized product is characterized by an opticaldensity of 1.8 and an MDH activity of 3 units/ml.

38. The process of claim 36 wherein said potassium phosphateconcentration is about 0.1 M.

39. The process of claim 35 wherein said buffer is a mixture ofpotassium phosphate and sodium phosphate.

40. The process of claim 39 wherein albumin is added in an amount toprovide a concentration of about 0.15 g./ 100 ml of reconstitutedlyophilized product.

41. A process for the preparation of a stable. lyophilized diagnosticcomposition as claimed in claim 9 which comprises admixing lactatedehydrogenase; a pyridine nucleotide selected from the group consistingof reduced nicotinamide adenine dinucleotide (NADH) and nicotinamideadenine dinucleotide phosphate (NADPH); and a buffer selected from thegroup consisting of potassium phosphate and a mixture of potassiumphosphate and sodium phosphate which regulates the pH of thereconstituted product in the range from 6 to 8; and lyophilizing theresulting aqueous product which upon reconstitutionis characterized byan optical density within the range from 2.1 to 1.0 acid and an LDHactivity of from 8 units/ml to about units/ml.

42. The process of claim 41 wherein said buffer is potassium phosphateadded as an 0.5 M aqueous solution whose concentration uponreconstitution is from 0.05 M to 0.4 M.

'43. The process of claim 41 wherein said pyridine nucleotide is NADHand the reconstituted lyophilized 11 12 product is characterized by anoptical density of 1.8 phate. and an LDH actlvlty 46. The process ofclaim 45 wherein albumin is 44. The process of claim 41 wherein sa1dbuffer 1s poadded in an amount to provide a concentration of tassiumphosphate and said potassium phosphate concentration is about 0.1 M.

45. The process of claim 41 wherein said buffer is a mixture ofpotassium phosphate and sodium phosabout 0.15 g./ 100 ml ofreconstituted lyophilized prod- 'g;;g UNITED STATES PATENT OFFICECERTIFICATE OF CORRECTION Patent No. 3,819,488 Dated June 25, 1974Inventor) Robert L. Rush, Luis P. Leon and Anne C. De Lea It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

Column 7, line 14, change "75:1" to 7.5:1

Column 8, line 6 change "Example IV" to Example III Signed and sealedthis 29th day of April 1975.

(SEAL) Attest:

c. MARSHALL DANN Commissioner -of Patents and Trademarks RUTH C. MASONArresting Officer

2. The composition as claimed in claim 1 wherein said buffer ispotassium phosphate present in an amount to provide a concentration offrom about 0.05 M to 0.4 M upon reconstitution.
 3. The composition asclaimed in claim 1 wherein said pyridine nucleotide is NADH.
 4. Thecomposition as claimed in claim 1 wherein said activity of MDH is about3 units/ml.
 5. The composition as claimed in claim 1 wherein saidoptical density is 1.8.
 6. The composition as claimed in claim 2 whereinsaid concentration is 0.1 M.
 7. The composition as claimed in claim 1wherein said buffer is a mixture of potassium phosphate and sodiumphosphate and albumin is included in an amount to provide aconcentration upon reconstitution of about 0.15 g./100 ml.
 8. A stableaqueous solution having a pH in the range from 6.0 to 8.0 whichcomprises the composition of claim 1 dissolved in water.
 9. A stable,lyophilized diagnostic composition effective in determining glutamicpyruvic transaminase (GPT) in a sample containing said enzyme whichconsists of lactate dehydrogenase (LDH) present in an amount to providean activity upon reconstitution of from 8 units/ml to about 80 units/ml;a pyridine nucleotide selected from the group consisting of reducednicotinamide adenine dinucleotide (NADH) and reduced nicotinamideadenine dinucleotide phosphate (NADPH) said pyridine nucleotide presentin an amount to obtain an optical density upon reconstitution in therange of from 2.1 to 1.0 spectrophotometrically determined at 340 nmusing a 10 mm light path; and a buffer selected from the groupconsisting of potassium phosphate and a mixture of potassium phosphateand sodium phosphate, said mixture having a sodium content less than 600meq./1 upon reconstitution, which regulates the pH of the reconstitutedproduct in the range from 6 to
 8. 10. The composition as claimed inclaim 9 wherein said buffer is potassium phosphate present in an amountto provide a concentration of from about 0.05 M to 0.4 M uponreconstitution.
 11. The composition as claimed in claim 9 wherein saidpyridine nucleotide is NADH.
 12. The composition as claimed in claim 9wherein said activity of LDH is about 40 units/ml.
 13. The compositionas claimed in claim 9 wherein said optical density is 1.8.
 14. Thecomposition as claimed in claim 10 wherein said concentration is 0.1 M.15. The composition as claimed in claim 9 wherein said buffer is amixture of potassium phosphate and sodium phosphate and albumin isincluded in an amount to provide a concentration upon reconstitution ofabout 0.15 g./100 ml.
 16. A stable aqueous solution having a pH in therange from 6.0 to 8.0 which comprises the composition of claim 9dissolved in water.
 17. In a method for quantitatively determining theactivity of glutamic oxalic transaminase (GOT) in a sample by mixingsaid sample and a substrate and analyzing for GOT; the improvement whichcomprises combining the stabilized composition as claimed in claim 1with the reaction product from said mixing step and photometricallymeasuring the decrease in NADH concentration.
 18. The method of claim 17including the further step of passing the reaction product through adialysis membrane prior to combining with said composition.
 19. Themethod of claim 17 including the further step of heating the reacTionmixture of said sample and substrate.
 20. The method of claim 17 whereinsaid substrate is an aspartic acid - Alpha -ketoglutaric acid mixture.21. The method of claim 17 wherein said composition buffer regulates thereconstituted product in the range from 6 to
 8. 22. The method of claim21 wherein said buffer is potassium phosphate.
 23. The method of claim21 wherein said buffer is a mixture of potassium phosphate and sodiumphosphate.
 24. The method of claim 22 wherein said composition comprisesmalate dehydrogenase, NADH and potassium phosphate.
 25. The method ofclaim 17 wherein said composition is in the form of an aqueous solutionhaving a pH in the range from 6.0 to 8.0.
 26. In a method forquantitatively determining the activity of glutamic pyruvic transaminase(GPT) in a sample by mixing said sample and a substrate and analyzingfor GPT, the improvement which comprises combining the stabilizedcomposition as claimed in claim 9 with the reaction product from saidmixing step and photometrically measuring the decrease in NADHconcentration.
 27. The method of claim 26 including the further step ofpassing the reaction product of said sample and said substrate through adialysis membrane prior to combining with said composition.
 28. Themethod of claim 26 including the further step of heating the mixture ofsaid sample and substrate.
 29. The method of claim 26 wherein saidsubstrate is an alanine - Alpha -ketoglutaric acid mixture.
 30. Themethod of claim 26 wherein said composition buffer regulates thereconstituted product in the range from 6 to
 8. 31. The method of claim30 wherein said buffer is potassium phosphate.
 32. The method of claim30 wherein said buffer is a mixture of potassium phosphate and sodiumphosphate.
 33. The method of claim 31 wherein said composition compriseslactate dehydrogenase, NADH and potassium phosphate.
 34. The method ofclaim 26 wherein said composition is in the form of an aqueous solutionhaving a pH in the range from 6.0 to 8.0.
 35. A process for thepreparation of a stable, lyophilized diagnostic composition as claimedin claim 1 which comprises admixing malate dehydrogenase, a pyridinenucleotide selected from the group consisting of reduced nicotinamideadenine dinucleotide (NADH) and nicotinamide adenine dinucleotidephosphate (NADPH); and a buffer selected from the group consisting ofpotassium phosphate and a mixture of potassium phosphate and sodiumphosphate which regulates the pH of the reconstituted product in therange from 6 to 8; and lyophilizing the resulting aqueous product whichupon reconstitution is characterized by an optical density within therange from 2.1 to 1.0 and an MDH activity of from 0.5 units/ml. to about10 units/ml.
 36. The process of claim 35 wherein said buffer ispotassium phosphate added as an 0.5 M aqueous solution whoseconcentration upon reconstitution is from 0.05 to 0.4 M.
 37. The processof claim 35 wherein said pyridine nucleotide is NADH and thereconstituted lyophilized product is characterized by an optical densityof 1.8 and an MDH activity of 3 units/ml.
 38. The process of claim 36wherein said potassium phosphate concentration is about 0.1 M.
 39. Theprocess of claim 35 wherein said buffer is a mixture of potassiumphosphate and sodium phosphate.
 40. The process of claim 39 whereinalbumin is added in an amount to provide a concentration of about 0.15g./100 ml of reconstituted lyophilized product.
 41. A process for thepreparation of a stable, lyophilized diagnostic composition as claimedin claim 9 which comprises admixing lactate dehydrogenase; a pyridinenucleotide selected from the group consisting of reduced nicotinamideadenine dinucleotide (NADH) and nicotinamide adenine dinucleotidephosphate (NADPH); and a buffer selected from the group consisting ofpotassium pHosphate and a mixture of potassium phosphate and sodiumphosphate which regulates the pH of the reconstituted product in therange from 6 to 8; and lyophilizing the resulting aqueous product whichupon reconstitution is characterized by an optical density within therange from 2.1 to 1.0 acid and an LDH activity of from 8 units/ml toabout 80 units/ml.
 42. The process of claim 41 wherein said buffer ispotassium phosphate added as an 0.5 M aqueous solution whoseconcentration upon reconstitution is from 0.05 M to 0.4 M.
 43. Theprocess of claim 41 wherein said pyridine nucleotide is NADH and thereconstituted lyophilized product is characterized by an optical densityof 1.8 and an LDH activity of 40 units/ml.
 44. The process of claim 41wherein said buffer is potassium phosphate and said potassium phosphateconcentration is about 0.1 M.
 45. The process of claim 41 wherein saidbuffer is a mixture of potassium phosphate and sodium phosphate.
 46. Theprocess of claim 45 wherein albumin is added in an amount to provide aconcentration of about 0.15 g./100 ml of reconstituted lyophilizedproduct.